Botulinum neurotoxins (BoNTs) are a dangerous bioterrorism threat due to their extreme potency and lethality, as well as their ease of production and transport. If untreated, poisoning by the BoNTs can progress to flaccid paralysis and death due to respiratory failure. However, timely post-exposure intervention can limit the effects of the circulating toxin. Our overall, long-term research objective is to generate a novel class of therapeutics that can be administered to individuals who have been poisoned by BoNT. Each BoNT is composed of a catalytic light chain whose entry into neurons is mediated by the heavy chain. Our strategy is based on the model that botulism-related flaccid paralysis is a downstream consequence of the zinc-dependent endopeptidase activity elaborated by the BoNT light chain. One of the most powerful approaches to inactivate the endopeptidase function of the BoNT light chains is rational design of inhibitors targeting the active site. To achieve this, we wilt combine computational and experimental approaches to develop lead inhibitor templates. In Specific Aim 1, we will use a powerful computational approach called dynamic pharmacophore modeling to identify computational leads to block the endopeptidase activities of the BoNTs. In this approach, the conformational flexibility of the protein and active site are taken into account through molecular dynamics simulations and the generation of a consensus, or dynamic, pharmacophore model using an ensemble of molecular dynamics-generated protein conformations. The dynamic pharmacophore model is then used to search databases of commercially available small molecules to generate computational lead compounds. In Specific Aim 2, we will test each computational lead for inhibitory activity using enzyme assays and in vitro cellular assays. A milestone of this work will be the identification of one or more lead inhibitor templates that block the action of wild type toxin using in vitro model systems. The results from this research will establish the groundwork and justification for future development and in vivo testing of these novel inhibitors using established animal models.